Urinary Extracellular Vesicles: Ultracentrifugation Method

Recently, urinary extracellular vesicles (EVs) have garnered interest as a potential source of noninvasive biomarkers of diseases related to urinary organs (kidney, bladder, urethra, and prostate).Ultracentrifugation is considered the gold standard method for isolation of EVs. However, the precipitates after ultracentrifugation steps are usually contaminated with soluble proteins, such as the Tamm–Horsfall protein (uromodulin).Therefore, ultracentrifugation on a sucrose–deuterium oxide (D2O) cushion for purer EV isolation is performed to remove these proteins. In addition, as a nonultracentrifugation method for EV isolation, we have also adopted the phosphatidylserine (PS) affinity method, which is a novel method for EV purification using the T-cell immunoglobulin domain and the mucin domain–containing protein 4 (Tim4).Here, we describe an ultracentrifugation protocol based on a sucrose–D2O cushion and the PS affinity method protocol for the isolation of urinary EVs.